Phenylalanine fluorescence and phosphorescence used as a probe of conformation for cod parvalbumin

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Phenylalanine fluorescence and phosphorescence used as a probe of conformation for cod parvalbumin
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  Journal of Fluorescence, VoL 3, No. 2, 1993 Phenylalanine Fluorescence and Phosphorescence Used as a Probe of Conformation for Cod Parvalbumin K. Sudhakar 1 W. W. Wright ~ S. A. Williams 2 C. M. Phillips 2 and J. M. Vanderkooi 1 ~ Received April 7, 1993; revised July 16, 1993 The fluorescence emission and triplet absorption properties of phenylalanine in cod fish parval- bumin type II, a protein that contains no Trp or Tyr, was examined in the time scale ranging from nanoseconds to microseconds at 25~ in aqueous buffer (pH 7.0). In the presence of Ca(II), the decay of fluorescence gave two lifetimes (5.9 and 53 ns) and the triplet lifetime was 425 txs. Upon removal of Ca, the fluorescence intensity decreased to values approaching that for free Phe, while the longest fluorescence decay component was 17 ns. At the same time, the decay of triplet showed complex nonexponential kinetics with decay rates faster than in the presence of Ca. Quenching and denaturation analyses suggest that the Phe's are present in a hydrophobic environment in the Ca-bound protein but that the Ca-free protein is relatively unstructured. It is concluded that Phe luminescence in proteins is sensitive to conformation and that the long lifetime of Phe excited states needs to be considered when studying its photochemistry in proteins. KEY WORDS: Parvalbumin; phenylalanine; fluorescence; phosphorescence. INTRODUC~ON Of all the amino acids in proteins, only the three aromatic amino acids, namely, phenylalanine, tyrosine, and tryptophan, exhibit fluorescence [1]. Whereas the fluorescence properties of Tyr and Trp are widely used to study proteins, the emission of Phe cannot be used to study proteins in most cases. Phe has a low molar ab- sorptivity (19.6 cm 2 mol-1) in comparison to Tyr (-180 cm 2 mol-1) or Trp (-580 cm 2 mo1-1) at their respective absorption maxima of 258, 275, and 280 nm. The quan- tum yield of Phe is low, and furthermore, its emission maximum at 282 nm overlaps the absorption of Tyr and i Johnson Research Foundation, Department of Biochemistry nd Bio- physics, School of Medicine, University of Pennsylvania, Philadel- phia, Pennsylvania 19104. 2 Regional Laser and Biotechnology Laboratory, Department of Chem- istry, University of Pennsylvania, Philadelphia, Pennsylvania 19104. 3 To whom correspondence hould be addressed. 57 Trp so that energy is effectively transferred to these moieties in proteins that contain them. These factors ex- plain why in all proteins and peptides, except those few that contain Phe but no Trp or Tyr, the emission of Phe is not even seen [2]. For the above reasons, the fluorescence of Phe has not been scrutinized in the detail given to Tyr or Trp. In spite of this, its excited-state properties deserve some attention. In a few proteins and polypeptides where Phe is the only aromatic amino acid, its fluorescence can serve as a useful marker. In addition, Phe may be in- volved in the UV photochemistry of proteins, and with the increasing flux of UV irradiation due to the depletion of the ozone layer, its photoreactions may have increas- ing biological significance. Proteins containing Phe, but devoid of both Tyr and Trp residues, occur rather seldom. One such protein ~s parvalbumin isozyme II from cod fish, which contair~s 10 Phe's but no Tyr's or Trp's [3]. It is a member of I053-0509/93/0300-0057507.00/0 9 1993 Plenum Publishing orporation  58 Sudhakar Wright Williams Phillips and Vanderkooi the family of parvalbumins, and while the role of par- valbumins in vertebrate muscle is not fully understood, they seem to be indirectly involved in the relaxation process of fast-twitch glycolytic fibers through their abil- ity to bind and release Ca(II) [4-7]. In this work, we studied the photophysical properties of Phe in parval- bumin II in Ca-bound and Ca-flee forms of the protein. MATERIALS AND METHODS Materials All chemicals were of chemically pure grade. Tris, EDTA, and guanidine hydrochloride (GHCI) were ob- tained from Sigma Chemical Co. (St. Louis, MO). Parvalbumin was prepared from frozen cod fillets obtained from the local supermarket. The procedure used slight modifications of published methods [3,8]. Frozen cod fillets (125 g) were homogenized in a blender with 200 ml of 10 mM Tris buffer (pH 8.7), 2 mM EDTA, and 2% glycerol, stirred for 1 h at 4~ and then cen- trifuged (16,300 g for 30 min). To the supernatant, ace- tone was added dropwise to give a 45% acetone (v/v) solution. It was then centrifuged for 75 rain at 16,300 g. The supernatant was taken to 80% acetone (v/v), the mixture was centrifuged, and the pellet was resuspended in 10-15 ml of 10 mM Tris buffer at pH 7.6 and 1 mM CaC12, heated rapidly (-5 min) to 60~ and then im- mediately cooled and centrifuged. Subsequent steps were at 4~ The supernatant was dialyzed against 1.6 mM piperazine at pH 5.7 overnight and then chromato- graphed (0.5 ml/min) on a Whatman DEAE-52 cellulose column (2.5 x 43 cm) equilibrated with the same buffer. The column was washed for -24 h with the piperazine buffer until the absorbance at 280 nm fell to N0.03 and then eluted with a NaC1 gradient (500 ml, 0-0.1 M NaC1, 0.5 ml/min). The eluted protein was detected by absor- bance at 280 and 260 nm. As shown in Fig. 1, peaks of the two parvalbumin species were separated; the peak eluting at 92 mM NaCI exhibits an absorbance maximum at 280 nm and is identified as the Trp-containing species. The next peak, with minimal absorbance at 280 nm and a maximum at about 260 nm, is identified as the protein containing the Phe residues but no Trp or Tyr. It eluted at -100 mM NaCI. Fractions between the arrows indi- cated in Fig. I were pooled, dialyzed against water, and lyophilized. Analysis of Denaturation Profile The procedure of Pace [9] was used to characterize the denaturation process of parvalbumin. Many globular 3.5 2,5 2 1,5 [I t, i ', 0 I b I 20 40 60 80 100 NaCI mM Fig. 1. Elution profile for DEAE cellulose as a function of salt gra- dient. The dashed line is the optical density at 280 nm; the solid line is the optical density at 260 nm. Fractions between the arrows were collected and used for this study. proteins have been found to approach closely a two-state mechanism: NoD (1) in which only the native state, N, and the denatured state, D, are present at significant concentrations in the transition region. The values fN and fD, the respective fraction of the protein present in the native and denatured states at different concentrations of GHC1, are obtained in the transition region by extrapolation from the linear portions of the denaturation curve at low and high den- aturant concentrations. AG is obtained from the follow- ing equation: AG = - 2.303 RT log fD/YN (2) Instrumentation Steady-state absorption spectra were obtained with a Model 200 Perkin-Elmer spectrometer. Steady-state fluorescence spectra were recorded on a Perkin-Elmer LS-5 luminescence spectrometer. Transient absorption spectra of the excited state species were acquired using the diode array instrument described in detail previously [10]. The actinic light, obtained from a Q-switched Nd:YAG laser, was 8 ns fwhm in duration and had a repetition rate of 10 Hz. The exciting wavelength was 266 nm. The instrument includes a Triplemate fiat-field spectrograph to resolve the spectrum and a Princeton Instruments DIDA-512 dual- diode array system to detect the spectrum. A xenon arc  Phe Fluorescence as a Probe for Cod Parvalbumin 59 lamp (Hammamatsu, Middlesex, NJ) provided probe light for photoexcited and unexcited regions of the sample. Baseline corrections and conversion of transmittance to absorbance were carried out by the computer software. This apparatus allows us to obtain the UV/VIS difference absorption spectrum over a range of 300 nm at variable times after the actinic pulse. The same instrument was also used for transient emission studies. The gate dura- tion for data accumulation was 5 ns, making the instru- ment suitable for emission studies in the submicrosecond time range and longer. For measuring the decay of emission on the nano- second time scale a time correlated single photon count- ing instrument, previously described [11], was used. The exciting light was a Coherent Antares modelocked YAG laser (Palo Alto, CA), which was quadrupled to give an excitation wavelength of 266 nm. Since the repetition rate of the pump source was 76 MHz, lifetimes in excess of 13 ns could not be measured with the modified in- strumental configuration and the relative amplitudes of short and long components cannot be accurately deter- mined. The decay of the fluorescence and of the triplet transient species were analyzed by the standard RLBL LIFETIME program (Holtom. 1989), which accounts for the instrument response function or by Excel (Microsoft Corporation, Redmond, WA), which did not deconvo- lute the instrument response. An exponential function was used to describe the decay of fluorescence, f, where the lifetime, % is given by N f(t) = ~ ai e-t/~i (3) i for i number of components (i = 1.0 for single exponen- tial) and A is the amplitude. Fluorescence Quenching The quenchability of the Phe fluorescence was ex- amined by monitoring fluorescence intensity, F, upon the addition of KI by this relationship [12]: Fo F = 1 + k'ro[KI ] (4) where/7o is the fluorescence intensity in the absence of quencher, 'r o is the fluorescence lifetime with no quencher, and k is the bimolecular quenching constant. The solutions of KI were prepared immediately be- fore use. A trace of sodium thiosulfate was added to the stock KI solutions to retard I3- formation. Oxygen Removal from Samples The buffer solution containing 0.3% glucose was initially degassed under an aspirator and then bubbled with argon. The protein was dissolved in the buffer and placed in a cuvette containing a glass-coated microstir bar, and the air space was filled with argon. A small volume of solution containing glucose oxidase and cat- alase was added to give a final concentration of 80 and 16 nM, respectively. This enzyme system catalyzed the reduction of 02 to H2Oz and then to H20. The cuvet~e was then closed with a quartz stopper. Throughout these operations, air was excluded by a constant flow of argon gas over the cuvette. Computer Graphics The coordinates for parvalbumin was obtained from the Brookhaven Data base, entry 5CPV, based upon co- ordinates results of Kumar et al. [13] and plotted using a Silicon Graphics computer. RESULTS Absorption and Emission Properties The UV absorption spectrum and fluorescence emission of the parvalbumin preparation used is shown in the Fig. 2. The fluorescence emission maximum is at 287 nm, which compares with 284 nm for Phe in water. The absorption maximum occurred at 259 nm; this wavelength did not detectably change between Phe in water and Phe in the protein environment (not shown). The fluorescence lifetime of Phe in parvalbumin has been reported to be nonsingle exponential with a short lifetime of 5.4 ns and a longer lifetime estimated to be 62 ns, but which could not be accurately determined with the then available instrumentation [14]. For the Ca-bound protein studied here, a fluorescence species with a life- time of 5.9 ns is seen using the time-correlated single photon counting apparatus (Fig. 3A). The data also had a contribution from a longer component of approxi- mately 50 ns. To determine the lifetime of the long component accurately, the fluorescence emission of Ca-bound par- valbumin was investigated using the transient absorp- tion/emission diode array spectrometer, Which has a time resolution >5 ns. The decay of fluorescence is shown in Fig. 3B; a fit to the curve gave a lifetime of 53 ns for the long-lived emitting component of protein in deox- ygenated buffer. Because two instruments were used to  60 Sudhakar Wright Williams Phillips and Vanderkooi lOO 80 9 60 40 20 0 ~ 240 260 280 300 320 340 Wavelength nm Fig. 2. Absorption (A) and fluorescence emission (B) spectra of par- valbumin. Medium contained 0.01 M Tris (pH 7.0) and 0.1 M NaCI. Excitation: 266 nm using a 3-nm effective bandpass. Temperature: 25~ 2500 200O ea lsoo .~ lOOO 500 L4- 1.2 1 0,8 0.6 0.4 ae 0.2 A \ i i : i I 1 2 3 4 5 6 Time, nsee B 50 100 150 200 250 300 Time nsec Fig. 3. Fluorescence decay using an emission wavelength of 295 nm and excitation at 266 nm. (A) Decay using the time-correlated single- photon counting nstrument. The solid line indicates an exponential it with a decay of 5.9 ns (,4 =1.31) and 53 ns (A=0.002). (B) Decay using the diode array nstrument. The solid ine shows an exponential fit with the lifetime of 53 ns. determine the lifetimes, the relative amplitudes of the long and short components was not obtained. The emission spectrum as a function of time was also recorded for the long-lived species. As shown in Fig. 4, the emission maximum remained at 287 nm, with no shifts in the emission spectra in the time scale of 15 to 100 ns. The emission in the range where phospho- rescence is expected to occur, i.e., 325 to 375 nm, did not decay to zero in the time scale examined, indicating that a longer-lived species is also emitting. Because of the limited gate time of the instrument a clear spectrum of the phosphorescence could not be obtained. At room temperature, the triplet state could be de- tected by its transient absorption using the diode array transient absorption spectrometer. The transient absorp- tion spectrum observed 1 p.s after excitation at 266 nm is shown in Fig. 5A. The spectrum was taken at different delay times, and while the absorbance decreased with increasing times, the spectrum showed no discernible shifts (not shown). The decay of the triplet in Ca-bound parvalbumin is shown in Fig. 5B; the lifetime was 425 ~s. The fluorescence and triplet absorption properties 400000 ! 5 nsec 300OOO -~ 2o00oo i 1o00oo ol 250 275 300 325 350 375 Wavelength am Fig. 4. Fluorescence emission spectra of Phe in cod parvalbumin, at the delay times indicated. Excitation wavelength, 266 nm; gate time, 5 ns. Samples contained 2 mg of protein/ml in 0.01 M Tris and 0.1 M NaC1 at pH 7.0. of Phe in the Ca-depleted parvalbumin and in free Phe were also examined. Addition of EDTA (2 raM) to the parvalbumin solution resulted in approximately 60% de-  Phe Fluorescence as a Probe for Cod Parvalbumin 61 0.08 0.06 Z 20.04 .< 0.02 0.00 A 250 300 350 400 450 WAVELENGTH, am 0.07 0.05 o ~0.02 "', 9 o ",, 0.01 o , "~" -n 0.01 0.1 1 10 100 1000 TIME, usec Fig. 5. (A) Transient absorption spectrum of Phe in cod parvalbumin after excitation at 266 nm. The delay time was 1 IXS and the gate time 5 ns. (B) Decay kinetics of triplet; plotted absorption maximum of parvalbumin (o) and Ca(II)-depleted parvalbumin (o) at -302 nm as a function of different delay times. The solid line is the simulated decay curve using the exponential 425 ~s with the absorbanee ampli- tude of 0.064 and the dashed line is the fit with absorbance amplitudes of 0.001, 0.025, and 0.03 and lifetimes of 0.09, 0.7, and 25 p.s. Samples were prepared by dissolving 2 mg parvalbumin/ml of 0.01 M Tris, 0.3% glucose, and 0.1 M NaC1 at pH 7.0. Deoxygenation was achieved using the enzyme system and other procedures described under Materials and Methods. crease in fluorescence intensity and a decrease the life- time of longer component from 53 to 17 ns. There was also a small blue shift (-2 nm) in the emission. The transient absorption spectrum of the excited triplet state species in the presence of EDTA was identical to that found for the Ca-bound parvalbumin, however, the de- cay of the triplet state was dramatically shortened. The absorbance as a function of delay time is also given in Fig. 5B for the Ca-depleted protein. The decay is no- nexponential. With regard to free Phe in aqueous solution, the transient triplet state lifetime is even further reduced. We found a decay time of 5 txs (data not shown), which compares well with the literature value for the triplet state lifetime of 3 ~s [15]. Sensitivity of Phe Fluorescence to Protein Conformation The decrease in fluorescence intensity and lifetime as well as the decrease in lifetime of the triplet state that was observed when Ca was removed from parvalbumin suggests that Ca removal results in exposure of Phe to the solvent. As a further indication of the exposure of Phe to solvent in the Ca-bound and Ca-free protein, quenching experiments were undertaken using KI. The data are shown in Fig. 6. The quenching constants can be determined from these data, using the fluorescence lifetimes. We used a lifetime of 5.9 ns for Phe in solution and 17 ns for Phe in the Ca-depleted protein and a life- time of 50 ns for the Ca-bound parvalbumin, the re- spective Stem-Volmer quenching constants of 63.6, 19.3, and 4.5 M-ins -1 for Phe in solution, for Ca(II)-depleted and Ca-bound parvalbumin. These values are approxi- mately in view that the relative amplitudes of short- and long-emitting species could not be determined with our instruments. Therefore, no attempt was made to distin- guish collisional and static effects [16]. The decrease in fluorescence intensity and in- creased susceptibility to quenching by I- upon removal of Ca suggests that the Phe becomes exposed to the solvent. If so, addition of a denaturant to unfold the protein should also result in a decrease in fluorescence intensity. The addition of GHC1 results in a decrease in fluorescence intensity (Fig. 7A) and a small (-2- to 3- 15 1 10- 9 5 A 9 0.00 0.;1 0.;2 0.;3 0.1~4 [KI], M Fig. 6. Stern-Volmer plot for quenching of parvalbumin (o), Ca-flee parvalbumin (A), and Phe (o) by KI. Parvalbumin, 1.4 mg/ml, or Phe, 10 g3,/, was dissolved in 0.01 M "Iris, pH 7.0, and 0.1 M NaC1. Ca(II) was removed by adding 5 ~ EDTA. Quenching was measured by the decrease in steady-state emission intensity, using 265 nm for excitation and 287 nm for emission.
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